Experiment 1: Study and plot the growth curve of E.

coli by turbidimetric and

standard plate count methods:

Introduction: Bacterial growth is defined as the increase in number of cells which occurs by cell
division (binary fission) according to a logarithmic progression pattern. Bacterial populations
grown in broth cultures follow a sequential series of four growth phases:

i) Lag phase ‐ The stage of bacterial growth where bacteria are acclimating to their new
environment and are gathering nutrients in readiness for cell division, increasing their size, and
synthesizing mainly enzymes.

ii) Log or exponential phase ‐ Here cells are duplicating at a constant rate and the cells are
metabolically active. This is the stage when the generation time of a culture can be determined.

iii) Stationary phase ‐ A stage when cell death equals cell growth. At this point in the growth
cycle there is no net increase in cell numbers. The medium at this stage contains limited nutrients
and the presence of toxic waste products generated from metabolism in large amounts.

iv) Death phase ‐ This stage is marked by the accumulation of toxic substances which results in
the decline of cell numbers. Many cells autolyse, and most cells have used up surrounding
nutrients.

When bacteria are inoculated into a liquid growth medium and the population is counted at
intervals, it is possible to plot a bacterial growth curve that shows the growth of cells over time.
The bacterial growth is determined spectrophotometrically or colorimetrically by determining the
turbidity.
Principle:

The bacterial growth rate is monitored by determining the increase in turbidity, because of
multiplication of cells during incubation, indicated by the increase in absorbance
spectrophotometrically or colorimetrically in comparison with the control (uninoculated broth).
The intensity of light striking the photodetector on the spectrophotometer or colorimeter is
inversely proportional to the number of bacteria.

Live cell densities can be measured by counting number of colonies after overnight incubation at
optimal growth temperature in solidified growth medium utilizing following formula‐ Colony
forming Units (CFU/ml) = No. of colonies × Dilution factor × Volume of culture plated

Requirements:

• Bacterial culture: overnight broth culture of E. coli (at least 100 ml of nutrient broth in a 500
ml sterile conical flask; inoculated with not more than 10 µl of bacterial suspension) grown at 37°C
in an incubator in shaking condition.

• Sterile nutrient broth: 25 ml in 100 ml side‐arm flask

. • Sterile test tubes

• Sterile Petriplates

• Sterile pipettes(1ml, 10ml)

• Colorimeter

• Incubator

• Sterile spreader

• Agar powder (for preparing nutrient agar to count live cell density)

Procedure:

• Nutrient Broth in side‐arm flasks heavily inoculated with overnight grown culture of E. coli In
duplicate (add about 5% of culture in each side-arm flask).
• O.D. at 600 nm measured in a colorimeter after calibration by an uninoculated sterile nutrient
broth medium in side‐arm flask (Blank). Average O.D. calculated. (This is the O.D. for t= 0 hr.)

Side‐arm flasks incubated at 37°C in shaking condition.

• At every 30 minutes interval, O.D.at 600 nm measured and average O.D. calculated.

• Live cell densities of for at least 6time points (taking at least 2 time points from each growth
phase) and 2 O.D after overnight incubation measured in solidified nutrient agar plates utilizing
the formula mentioned in the Principle section. Preferably serial dilution upto appropriate level to
be made before pour or spread plating in aseptic condition.

• Two graphs prepared‐ one for average O.D.600 Vs. Time and the other for average log
Special points for discussion:

• For unicellular organisms, OD is proportional, within certain limits, to cell number. Turbidity
readings can therefore be used as a substitute for total or viable counting methods. However, before
this can be done, a standard curve must be prepared that relates cell number microscopic or viable
count), dry weight, or protein content to turbidity. As can be seen in such a plot, proportionality
only holds within limits. Thus, at high cell concentrations, light scattered away from the
spectrophotometer’s photocell by one cell can be scattered back by another. To the photocell, this
is as if light had never been scattered in the first place. At such high cell densities, the one‐to‐one
correspondence between cell number and turbidity deviates from linearity, and OD measurements
become less accurate. However, up to this limit, turbidity measurements can be accurate measures
of cell number or dry weight.

• Also, because different organisms differ in size and shape, equal cell numbers of two different
bacterial species will not necessarily yield the same OD. Thus, to relate OD to actual cell numbers,
a standard curve relating the set two parameters must be made for each different organism grown
routinely in the laboratory.

• Turbidity measurements have the virtue of being quick and easy to perform. Turbidity
measurements can typically be made without destroying or significantly disturbing the sample. For
these reasons, turbidity measurements are widely employed to monitor growth of microbial
cultures. The same sample can be checked repeatedly and the measurements plotted on a semi
logarithmic plot versus time. From these, it is easy to calculate the generation time and other
parameters of the growing culture.

• Turbidity measurements are sometimes problematic. Although many microorganisms grow in

even suspensions in liquid medium, many others do not. Some bacteria form small to large clumps,
and in such instances, OD measurements may be quite inaccurate as a measure of total microbial
mass. In addition, many bacteria grow in films on the sides of tubes or other growth vessels,
mimicking in laboratory culture how they actually grow in nature. Thus for ODs to accurately
reflect cell mass (and thus cell numbers) in a liquid culture, clumping and biofilms have to be
minimized. This can often be accomplished by stirring, shaking, or in some way keeping the cells
well mixed during the growth process to prevent the formation of cell aggregates and biofilms.